Vaccination elicits high affinity antibodies to pathogens and toxins to protect the individual. While T cells recognize antigen fragments with a fixed, low affinity, B cells initially generate low affinity antibodies and then undergo a process of focused hypermutation and selection to generate high affinity antibodies. The vital process of mutation and selection takes place in germinal centers (GCs), highly organized foci of antigen specific B cells within B follicles of the secondary lymphoid tissues. Experiments over the past 40 years have shown that GC contain two zones- the light zone in which B cells that have mutated their antibody genes by expressing activation-induced cytidine deaminase (AID)are selected by antigen to produce high affinity antibodies and a dark zone in which selected B cells proliferate. The selected and expanded B cells then differentiate into antibody producing plasma cells or memory B cells. The precise mechanism by which high affinity B cells are selected from a pool of B cells with diverse affinities is still unclear. Follicular dendritic cells (FDCs) found primarily in the light zone present intact antigens to B cells in the form of immune complexes. It has been proposed that B cells compete for antigen provided by FDCs and then present these antigens to follicular T cells to obtain survival and differentiation signals. The goal of the proposed work is to elucidate these mechanisms by investigating the cellular interactions of antigen specific B cells with T cells, FDCs and DCs during the GC reaction using a combination of molecular genetics and two-photon laser scanning intravital microscopy (TPLSIM), a method for submicron fluorescence imaging relatively deep in tissues (up to 400 mm). This will be a collaborative effort between the Nussenzweig and Dustin laboratories located at The Rockefeller University and NYU Medical School. Transgenic B- and T- cells expressing defined antigen receptor specificities, bone marrow-derived DCs and FDCs will be labeled with different fluorescent protein markers. In addition, antigen and immune complexes will be tagged with fluorescent dyes or semiconductor nanocrystals (Quantum dots[unreadable]). In the first Aim we will study the interactions of antigen specific B cells with antigen, Th cells and FDCs, and examine competition between low and high affinity B cells. In Aim 2 we will use genetic tagging methods to study endogenous antibody responses and define the molecular mechanisms that produce light and dark zones and govern the entry and exit of follicular B cells into GCs. These studies will be directly applicable to optimizing vaccination strategies for high affinity antibody production.